Paclitaxel Induces Micronucleation and Activates Pro-Inflammatory cGAS-STING Signaling in Triple-Negative Breast Cancer

Abstract

Taxanes remain one of the most effective medical treatments for breast cancer. Clinical trials have coupled taxanes with immune checkpoint inhibitors in patients with triple-negative breast cancer (TNBC) with promising results. However, the mechanism linking taxanes to immune activation is unclear. To determine if paclitaxel could elicit an antitumoral immune response, we sampled tumor tissues from patients with TNBC receiving weekly paclitaxel (80 mg/m²) and found increased stromal tumor-infiltrating lymphocytes and micronucleation over baseline in three of six samples. At clinically relevant concentrations, paclitaxel can induce chromosome missegregation on multipolar spindles during mitosis. Consequently, post-mitotic cells are multinucleated and contain micronuclei, which often activate cyclic GMP-AMP synthase (cGAS) and may induce a type I IFN response reliant on the stimulator of IFN genes (STING) pathway. Other microtubule-targeting agents, eribulin and vinorelbine, recapitulate this cGAS/STING response and increased the expression of immune checkpoint molecule, PD-L1, in TNBC cell lines. To test the possibility that microtubule-targeting agents sensitize tumors that express cGAS to immune checkpoint inhibitors, we identified 10 patients with TNBC treated with PD-L1 or PD-1, seven of whom also received microtubule-targeting agents. Elevated baseline cGAS expression significantly correlated with treatment response in patients receiving microtubule-targeting agents in combination with immune checkpoint inhibitors. Our study identifies a mechanism by which microtubule-targeting agents can potentiate an immune response in TNBC. Further, baseline cGAS expression may predict patient treatment response to therapies combining microtubule-targeting agents and immune checkpoint inhibitors.

Fig. 1.. Some but not all triple-negative breast cancer patients develop tumor-infiltrating lymphocytes following neoadjuvant paclitaxel.

(A) (Above) Dose scheduling of patients with biopsies (P102, P103, P113, P114, P118, P119) undergoing neoadjuvant paclitaxel treatment. Treatment regimen comprises biweekly 80mg/m² neoadjuvant paclitaxel for four doses. (Below) Representative hematoxylin and eosin (H&E) images showing increased stromal tumor-infiltrating lymphocytes (sTILs) in one patient P102 after the third dose of 80mg/m² neoadjuvant paclitaxel 15 days after the start of therapy. Inset contains magnifications of representative patient images. Tumor is outlined in black and sTILs are represented by small cells with dark, purple nuclei in “Day 15” sample. Original images are at 400x magnification. (B) Quantification of sTILs of patient H&E images by staff pathologist using International TILs Working Group criteria. See supporting information for more details. Qualitative TILs categories are A=low (0–20%), B=medium (20–50%), C=high (50–100%). Red shading represents patients with TILs increased to a higher qualitative category at “Day 15” compared to baseline. Blue shading represents patients with TILs that have either not increased or have not increased to a higher qualitative category. (C) Representative images of tumor cells from patient biopsies of nuclear phenotypes after paclitaxel showing DNA (DAPI), cGAS and pan-cytokeratin (PCK). Micronucleus is represented by yellow arrow and magnified in the inset panels on the bottom-right corner. Scale bar, 50μm. (D) Quantification of intratumoral cGAS expression by average (mean) cGAS signal intensity of tumor cell clusters. Red shading represents patients with TILs increased to a higher qualitative category at “Day 15” compared to baseline. Blue shading represents patients with TILs that have either not increased or have not increased to a higher qualitative category. AU=arbitrary units (E) Spearman correlation of six patients treated with neoadjuvant paclitaxel from (A-D) plotted by change in TILs between “Day 15” and “Baseline”. Red squares represent patients with TILs increased to a higher qualitative category at “Day 15” compared to baseline. Blue squares represent patients with TILs that have either not increased or have not increased to a higher qualitative category. AU=arbitrary units

Fig. 2.. Paclitaxel causes cGAS-positive micronuclei to form in interphase following delayed mitosis on multipolar spindles.

(A) Representative images of MDA-MB-231 cells expressing H2B-RFP and tubulin-GFP filmed with 60X objective from mitosis to subsequent interphase with no treatment (UNT) at 5-min intervals or 10 nM paclitaxel (PAX) at 4-min intervals. (B) Quantification of live cell images from (A) for mitotic duration and chromosome segregation defects from Nuclear Envelope Breakdown (NEBD) to anaphase onset and daughter cell interphase nucleus morphology. N=50 cells for UNT and N=150 cells for 10 nM PAX. (C) Representative images of multinucleated MDA-MB-231 after 10 nM paclitaxel with and without cGAS puncta. Scale bar, 10 μm. (D) Quantification of nuclei phenotypes (sum of blue and green bars) and cGAS-positivity (green bar) in MDA-MB-231 interphase cells after 0, 24, 48 and 72 hours of 10 nM paclitaxel. N=3 independent experiments comprising ≥250 asynchronous cells per condition. Mean and SEM are plotted. P values are calculated by one-way ANOVA with Tukey-Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001, ns=not significant). See Figure S2A for representative images of nuclei phenotypes. (E) Quantification of micronuclei (sum of blue and green bars) of multinucleated cells of multiple TNBC cell lines after 0 and 48 hours of 10 nM paclitaxel N=3 independent experiments comprising ≥250 asynchronous cells per condition. Results for MDA-MB-231 from (D). Mean and SEM are plotted. P-values were calculated by two-tailed, paired Student’s T Test (*P < 0.05, **P < 0.01, ***P < 0.001, ns=not significant). TNBC subtypes are M=mesenchymal, MSL=mesenchymal stem-like (retired classification), BL1=basal-like 1, BL2=basal-like 2, LAR=luminal androgen receptor. (F) Representative images of multinucleated MDA-MB-231 after 10 nM paclitaxel stained for cGAS and CREST. Yellow arrows indicate a cGAS-positive micronucleus with one CREST foci, which is magnified in the top-left inset. Scale bar, 10 μm. (G) Quantification of MDA-MB-231 CREST foci with no treatment (UNT) or after two days of 10 nM paclitaxel (10 nM PAX) categorized by number of cGAS-positive or cGAS-negative micronuclei. N=3 independent experiments comprising ≥50 asynchronous cells (≥ total 100 micronuclei) per condition. Mean and SEM are plotted. P values were calculated by one-way ANOVA with Tukey-Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001, ns=not significant).

Fig. 3.. Paclitaxel activates cGAS signaling in triple-negative breast cancer cell lines.

(A) ELISA analysis of 2’3’-cGAMP production in wild type and cGAS knockout MDA-MB-231 four days after exposure to 10 nM paclitaxel. N=3 independent experiments. Mean and SEM are plotted. P values were calculated by one-way ANOVA with Dunnett’s test with control as 0 day (*P < 0.05, **P < 0.01, ***P < 0.001, ns=not significant). (B) Representative immunoblot of MDA-MB-231 wild type cells exposed to 10 nM paclitaxel after 0, 0.5, 1, 2 and 4 days probed with phosphoproteins downstream of the STING pathway that increase with activation of the pathway comprising pTBK1, pSTING, pIRF3 and pSTAT1. (C) Representative immunoblot of cGAS, STING and pIRF3 from MDA-MB-231 wild type (WT) and cGAS knockout (KO) cells exposed to 10 nM paclitaxel for four days. Alpha tubulin serves as the loading control. (D) Representative immunoblot of cGAS and pIRF3 from MDA-MB-436 wild type (WT) and siRNA-mediated cGAS knockdown (KD) cells exposed to 10 nM paclitaxel for four days. Alpha tubulin serves as the loading control. (E) Interferon-beta measurements of wild type (WT) and cGAS knockout (KO) MDA-MB-231 exposed to no treatment (UNT) or 10 nM paclitaxel (PAX) over five days. N=2 independent experiments. Mean and SEM are plotted. P values were calculated by one-way ANOVA with Tukey-Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001, ns=not significant). (F) Interferon-beta measurements of MDA-MB-436 wild type (WT) and siRNA-mediated cGAS knockdown (KD) exposed to no treatment (UNT) or 10 nM paclitaxel (PAX) over five days. N=2 independent experiments. Mean and SEM are plotted. P values were calculated by one-way ANOVA with Tukey-Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001, ns=not significant).

Fig. 4. Paclitaxel induces secretion of cGAS-dependent soluble factors in breast cancer cells that polarize macrophages to an M1-like phenotype.

(A) Schematic of mono-culture conditioned media experiments with macrophages derived from PBMCs. (B) Representative images of human macrophages expressing CD80 incubated with conditioned media from MDA-MB-231 parental and cGAS knockout cell lines untreated or pre-treated with 10 nM paclitaxel. (C) Measurements of CD80 expression on macrophages following conditioned media from parental and cGAS knockout MDA-MB-231 untreated or pre-treated with paclitaxel. N=3 independent experiments. Each dot represents one cell. Mean and SEM are plotted. P values were calculated by one-way ANOVA with Tukey-Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001, ns=not significant). (D) Measurements of greatest dimension of macrophages following conditioned media from parental and cGAS knockout MDA-MB-231 untreated or pre-treated with paclitaxel. N=3 independent experiments. Each dot represents one cell. Mean and SEM are plotted. P values were calculated by one-way ANOVA with Tukey-Kramer test (*P < 0.05, **P < 0.01, ***P < 0.001, ns=not significant).

Fig. 5.. Nanomolar paclitaxel and other microtubule-targeting agents can increase surface PD-L1 expression in a cGAS-dependent manner in MDA-MB-436.

(A) Representative immunoblots of total PD-L1 expression in wild type and si-cGAS MDA-MB-436 cells before and after three days of 10 nM paclitaxel. Alpha tubulin serves as the loading control. (B) Flow cytometry strategy for PD-L1 expression. (C) Quantification of surface PD-L1 by percent of the live parent population of MDA-MB-436 cells, wild type and siRNA-mediated cGAS knockdown, before and after three days of 10 nM paclitaxel. Mean and SEM of n=3 independent biological experiments comprising ≥100,000 events per condition are shown. P values were calculated by one-way ANOVA with Dunnett’s test with control as 0 day (*P < 0.05, **P < 0.01, ***P < 0.001, ns=not significant). (D) Quantification of surface PD-L1 by percent of the live parent population of wild type MDA-MB-436 cells before and after three days of 10 nM eribulin and vinorelbine. Mean and SEM of n=3 independent biological experiments comprising ≥100,000 events per condition are shown. P values were calculated by one-way ANOVA with Dunnett’s test with control as 0 day (*P < 0.05, **P < 0.01, ***P < 0.001, ns=not significant).

Fig. 6.. Patients expressing higher levels of cGAS have increased disease control after combined microtubule-targeting agents and PD-1/PD-L1 inhibitors.

(A) Representative images of patient tumors from diagnostic biopsies with DNA (DAPI), cGAS and pan-cytokeratin (PCK) staining shown. Representative images by order of appearance from top to bottom are: patient sample with a high level of cGAS staining (cGAS^high), patient sample low level of cGAS staining (cGAS^low), background control with only secondary antibody and no primary antibody staining (−1°Ab), positive control for cGAS derived from paraffin-embedded MDA-MB-231 cell pellet (MDA-231), and negative control for cGAS derived from HEK293T cell pellet (HEK-293T). Scale bar, 50 μm. (B) Quantification of intratumoral cGAS expression by average (mean) cGAS signal intensity of tumor cell clusters. AU=arbitrary units (C) Immunoblot of cGAS in MDA-MB-231 and HEK293T, which serve as positive and negative controls for cGAS staining. (D) Swimmer’s plot showing progression free survival of patients in months after combination therapy of paclitaxel and atezolizumab (PD-L1) or eribulin and pembrolizumab (PD-1) stratified from top to bottom by high to low cGAS expression. Legend indicates treatment regimen (top) and response status (bottom). Top legend color coding also applies to (E) for patient treatments. (E) Spearman correlation of seven patients treated with both microtubule and immune checkpoint inhibitors from (B-C) plotted by mean cGAS intensity against progression free survival in months. This analysis excludes three patients without microtubule inhibitor treatment. AU = Arbitrary Units